A model for enzyme-substrate interaction in alanine racemase.

We report on a theoretical model for the complex of the enzyme alanine racemase with its natural substrate (L-alanine) and cofactor (pyridoxal 5'-phosphate). Electrostatic potentials were calculated and ionization states were predicted for all of the ionizable groups in alanine racemase. Some rather unusual charge states were predicted for certain residues. Tyr265' has an unusually low predicted pK(a) of 7.9 and at pH 7.0 has a predicted average charge of -0.37, meaning that 37% of the Tyr265' residues in an ensemble of enzyme molecules are in the phenolate form. At pH 8-9, the majority of Tyr265' side groups will be in the phenolate form. This lends support to the experimental evidence that Tyr265' is the catalytic base involved in the conversion of L-alanine to D-alanine. Residues Lys39 and Lys129 have predicted average charges of +0.91 and +0.14, respectively, at pH 7.0. Lys39 is believed to be the catalytic base for the conversion of D-alanine to L-alanine, and the present results show that, at least some of the time, it is in the unprotonated amine form and thus able to act as a base. Cys311', which is located very close to the active site, has an unusually low predicted pK(a) of 5.8 and at pH 7.0 has a predicted average charge of -0.72. The very low predicted charge for Lys129 is consistent with experimental evidence that it is carbamylated, since an unprotonated amine group is available to act as a Lewis base and form the carbamate with CO(2). Repeating the pK(a) calculations on the enzyme with Lys129 in carbamylated form predicts trends similar to those of the uncarbamylated enzyme. It appears that the enzyme has the ability to stabilize negative charge in the region of the active site. Implications for selective inhibitor design are discussed.